Biochemical Analysis

Total DNA, RNA and protein were isolated from L. casei BL23 microvesicles using 1 ml of Quick-Zol (Kalium Technologies) reagent following the manufacturer’s instructions. DNA and RNA were quantified by UV absorbance using a NanoDrop 2000/2000c (Thermo Fisher Scientific, United States). The protein content was quantified by Lowry’s method (Lowry et al., 1951). The reported values correspond to the average and standard deviation of three measurements.

Proteins were separated on a SDS-PAGE gel (12% resolving gel) followed by Coomassie blue staining (n = 5). Extracted proteins of MVs and bacteria were digested with trypsin and analyzed by nano-HPLC coupled to mass spectrometry with Orbitrap technology (LC-MS) (Wada et al., 2014). The protein spectrum was analyzed against L. casei BL23 proteome obtained from the genome sequence ({"type":"entrez-nucleotide","attrs":{"text":"NC_010999.1","term_id":"191636824","term_text":"NC_010999.1"}}NC_010999.1) (Mazé et al., 2010). Data were filtered using the Proteome Discoverer 2.1 software to obtain maximum protein and peptide false discovery rate of 1% calculated by employing a reverse database strategy. Raw data was deposited in EVpedia (evpedia.info/) (Kim et al., 2015).